Peptides which effect release of hormones

ABSTRACT

The present invention relates to peptides which possess biological activity in respect to the inhibition of growth hormone, insulin secretion and glucagon secretion are provided. The peptides have fewer amino acid components than somatostatin and some of the peptides have dissociated activity.

The present invention relates generally to peptides having biologicalactivity in respect to the inhibition of growth hormone, insulin andglucagon secretion. More particularly, the present invention is directedto peptides having fewer amino acid moieties than somatostatin which areeffective to inhibit the release of growth hormone by the pituitarygland or the release of glucagon or insulin by the pancreas. Variouspeptides of the invention have dissociated biological activity inrespect to the inhibition of growth hormone, insulin and glucagonsecretion.

A peptide having inhibitory effect on the secretion of growth hormonehas been characterized and is described in U.S. Pat. No. 3,904,594 toGuillemin et al. This peptide has been named "somatostatin".Somatostatin (also known as somatotropin release inhibiting factor) isthe tetradecapeptide: ##STR1##

Somatostatin, the linear form of somatostatin (dihydrosomatostatin) andvarious acylated derivatives of somatostatin and dihydrosomatostatin aredescribed in the aforementioned U.S. patent.

Somatostatin and many analogs of somatostatin exhibit activity inrespect to the inhibition of growth hormone (GH) secretion fromcultured, dispersed, rat anterior pituitary cells in vitro andinhibition of insulin and glucagon secretion in vivo in the rat. It hasbeen considered highly desirable in the use of somatostatin toselectively inhibit only the secretion of GH, insulin or glucagon.Efforts have been made to develop analogs of somatostatin which possessdissociated biological activity and which inhibit only GH, insulin orglucagon secretion. Although there have been reports citing differencesin the amounts of somatostatin required for inhibition of insulincompared to glucagon in the human and the perfused rat pancreas invitro, somatostatin and some somatostatin analogs exhibit similarpotencies on the inhibition of these two hormones in vivo.

The present invention relates to the discovery that certain amino acidscan be removed and/or rearranged in somatostatin and dihydrosomatostatinpeptides to provide novel peptides having fewer amino acid componentsand which possess biological activity in respect to the inhibition ofGH, insulin or glucagon secretion. Some of the novel peptides of theinvention have dissociated activity. The novel peptides of the inventionhaving fewer amino acid components than somatostatin ordihydrosomatostatin are considered to be of great value because of therelative simplicity with which these peptides can be manufactured.

The novel peptides of the invention are defined by the formulae:

    Cys-R.sub.1 -Phe-Phe-R.sub.2 -Lys-R.sub.3 -Phe-R.sub.4 -R.sub.5 -Cys I ##STR2## where R.sub.1 is selected from Asn and des R.sub.1, R.sub.2 is selected from Trp and D-Trp, R.sub.3 is selected from Phe and Thr, R.sub.4 is selected from Thr and des R.sub.4, and R.sub.5 is selected from Ser, Phe, and des R.sub.3 provided that at least one of R.sub.1, R.sub.4 and R.sub.5 is deleted.

The nomenclature used to describe the peptides of the present inventionis in accordance with the conventional practice of using the first threeletters of the trivial name. Also, in accordance with such practice, itis the L form of the amino acid that is intended, unless otherwiseexpressly indicated. In this connection, it should be understood thateither of the Cys amino acid moieties can be either D-Cys or L-Cys.

Pharmaceutically acceptable acid addition salts of the peptides are alsowithin the scope of the present invention. Such acid addition saltsinclude but are not limited to hydrochloride, hydrobromide, sulfate,phosphate, maleate, acetate, citrate, benzoate, succinate, malate,ascorbate, tartrate and the like.

Also considered to be within the scope of the present invention areintermediates of the formula:

    X-Cys(X.sup.1)-R.sub.1 -Phe-Phe-R.sub.2 -Lys(X.sup.2)-R.sub.3 (X.sup.3)-Phe-R.sub.4 (X.sup.4)-R.sub.5 (X.sup.5)- Cys(X.sup.6)-R.sub.6. III

wherein: X is either hydrogen or an α-amino protecting group. Theα-amino protecting groups contemplated by X are those known to be usefulin the art in the step-wise synthesis of polypeptides. Among the classesof α-amino protecting groups covered by X are (1) acyl type protectinggroups such as formyl, trifluoroacetyl, phthalyl, toluenesulfonyl(tosyl), benzensulfonyl, nitrophenylsulfenyl, tritylsulfenyl,o-nitrophenoxyacetyl, chloroacetyl, acetyl, y-chlorobutyrul, etc.; (2)aromatic urethan type protecting groups such as benzyloxycarbonyl andsubstituted benzyloxycarbonyl such as p-chlorobenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,p-methoxybenzyloxycarbonyl; (3) aliphatic urethan protecting groups suchas α-t-butyloxycarbonyl, diisopropylmethoxycarbonyl,isopropyloxycarbonyl, ethoxycarbonyl, allyloxycarbonyl; (4) cycloalkylurethan type protecting groups such as cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl; (5) thiourethan typeprotecting groups such as phenylthiocarbonyl; (6) alkyl type protectinggroups such as triphenylmethyl (trityl), benzyl; (7) trialkylsilanegroups such as trimethylsilane. The preferred α-amino protecting groupdefined by R is tertbutyloxycarbonyl.

X¹ and X⁶ are each a protecting group for Cys selected from the groupconsisting of S-p-methoxybenzyl, S-p-methylbenzyl, S-acetamidomethyl,S-trityl, S-benzyl, and the like. The preferred protecting group isS-p-methoxybenzyl. X¹ and/or X⁵ can be hydrogen which means that thereis no protecting group on the sulfur group.

X² is a protecting group for the side chain amino substituent of lysineor X² is hydrogen which means there is no protecting group on the sidechain amino substituent. Illustrative of suitable side chain aminoprotecting groups are benzyl, chlorobenzyloxycarbonyl,benzyloxycarbonyl, tosyl, t-amyloxycarbonyl, t-butyloxycarbonyl, etc.The selection of such a side chain amino protecting group is notcritical except that it must be one which is not removed duringdeprotection of the α-amino groups during the synthesis. Hence, theα-amino protecting and side chain amino protecting group cannot be thesame.

X³, X⁴ and X⁵ are protecting groups for the hydroxyl group of Thr andSer and are selected from the group consisting of acetyl, benzoyl,tert-butyl, trityl, tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl andbenzyloxycarbonyl. The preferred protecting group is benzyl. X³ and/orX⁴ and/or X⁵ can be hydrogen which means there is no protecting group onthe hydroxyl group.

R₁, R₂, R₃, R₄ and R₅ are as previously defined. R₆ is selected from theclass consisting of OH, OCH₃, esters, amides, hydrazides and benzylester or hydroxymethyl ester anchoring bond used in solid phasesynthesis linked to a solid resin support represented by the formulae:

    --O--CH.sub.2 -polystyrene resin support

and

    O--CH.sub.2 -benzyl-polystyrene resin support

The polymer is preferably a copolymer of styrene with about 0.5 to 2.0%divinyl benzene as a cross-linking agent which causes the polystyrenepolymer to be completely insoluble in certain organic solvents. Informula III at least one of X, X¹, X², X³, X⁴, X⁵ and X⁶ is a protectinggroup.

In selecting a particular side chain protecting group to be used insynthesis of the peptides of formula I or formula II, the followingrules should be followed: (a) the protecting group must be stable to thereagent and under the reaction conditions selected for removing theα-amino protecting group at each step of the synthesis, (b) theprotecting group must retain its protecting properties and not be splitoff under coupling conditions, and (c) the side chain protecting groupmust be removable upon the completion of the synthesis containing thedesired amino acid sequence under reaction conditions that will notalter the peptide chain.

The peptides of formula I and formula II can be prepared using solidphase synthesis. The synthesis is commenced from the C-terminal end ofthe peptide using an α-amino protected resin. Such a starting materialcan be prepared by attaching an α-amino and S-protected Cys to achloromethylated resin or a hydroxymethyl resin. The preparation of thehydroxymethyl resin is described by Bodanszky et al., Chem. Ind.(London) 38, 1597-98 (1966). A chloromethylated resin is commerciallyavailable from Bio Rad Laboratories, Richmond, California and thepreparation of such a resin is described by Stewart et al., "Solid PhasePeptide Synthesis" (Freeman & Co., San Francisco 1969), Chapter 1, pp1-6. The α-amino and S-protected Cys is coupled to the chloromethylatedresin according to the procedure of Monahan and Gilon, Biopolymer 12, pp2513-19, 1973. Following the coupling of the α-amino and S-protected Cysto the resin support, the α-amino protecting group is removed such as byusing trifluoroacetic acid in methylene chloride, trifluoroacetic acidalone or HCl in dioxane. The deprotection is carried out at atemperature between about 0° C. and room temperature.

Other standard cleaving reagents and conditions for removal of specificα-amino protecting groups may be used as described in Schroder & Lubke,"The Peptides", 1 pp 72-75 (Academic Press 1965).

After removal of the α-amino protecting group of Cys the remainingα-amino and side chain protected amino acids are coupled step-wise inthe desired order to obtain a compound of formula III or as an alternateto adding each amino acid separately to the synthesis, some of them maybe coupled prior to addition to the solid phase reactor. The selectionof an appropriate coupling reagent is within the skill of the art.Particularly suitable as a coupling reagent is N,N¹ -dicyclohexylcarbodiimide.

The activating reagents used in the solid phase synthesis of thepeptides are those well known in the peptide art. Examples of suitableactivating reagents are: (1) carbodiimides such as N,N-diisopropylcarbodiimide, N-ethyl N¹ -(y-dimethylamino propyl carbodiimide); (2)cyanamides such as N,N-dibenzylcyanamide; (3) keteimines; (4)isoxazolium salts such as N-ethyl-5-phenyl isoxazolium-3¹ -sulfonate;(5) monocyclic nitrogen containing heterocyclic amides of aromaticcharacter containing one through 4 nitrogens in the ring such asimidazolides, pyrazolides, 1,2,4-triazolides. Specific heterocyclicamides that are useful include N,N¹ -carbonyl diimidazole, N,N¹-carbonyl-di-1,2,4-triazole; (6) alkoxylated acetylene such asethoxyacetylene; (7) reagents which form a mixed anhydride with thecarboxyl moiety of the amino acid such as ethylchloroformate andisobutylchloroformate and (8) nitrogen-containing heterocyclic compoundshaving a hydroxy group on one ring nitrogen such asN-hydroxyphthalimide, N-hydroxysuccinimide and 1-hydroxybenzotriazole.Other activating reagents and their use in peptide coupling aredescribed by Schroder & Lubke supra, in Chapter III and by Kapoor, J.Pharm. Sci., 59, pp 1-27 (1970).

Each protected amino acid or amino acid sequence is introduced into thesolid phase reactor in about a fourfold excess and the coupling iscarried out in a medium of dimethylformamide: methylene chloride (1:1)or in dimethylformamide: methylene chloride (1:1) or indimethylformamide or methylene chloride alone. In cases where incompletecoupling occurred the coupling procedure is repeated before removal ofthe α-amino protecting group, prior to the coupling of the next aminoacid. The success of the coupling reaction at each stage of thesynthesis is monitored by the ninhydrin reaction, as described by E.Kaiser et al., Analyt. Biochem, 34, 595 (1970).

After the desired amino acid sequence of formula III has beensynthesized, the peptide is removed from the resin support by treatmentwith a reagent such as liquid hydrogen fluoride which not only cleavesthe peptide from the resin but also cleaves all remaining side chainprotecting groups X¹, X², X³, X⁴, X⁵ and X⁶ and the α-amino protectinggroup X to obtain directly a peptide of formula I. Peptides inaccordance with formula II are obtained by oxidizing formula I peptidesin accordance with known procedures. As an alternate route, the peptidelinked to the resin support may be separated from the resin byalcoholysis after which the recovered C-terminal methyl ester isconverted to the acid by hydrolysis. Any side chain protecting group maythen be cleaved as previously described or by other procedures such ascatalytic reduction (e.g. Pd on BaSO₄) using conditions which will keepthe Trp moiety intact. When using hydrogen fluoride for cleaving,anisole is included in the reaction vessel as a scavenger.

The solid phase synthesis procedure discussed above is well known in theart and has been essentially described by Merrifield J. Am. Chem. Soc.,85, p 2149 (1964).

The peptides of the present invention having dissociated effects inrespect to inhibition of release of growth hormone, insulin and glucagonare considered to be particularly important in connection with thetreatment of diabetes. The traditional view of diabetes has been that itis a disease resulting from impaired insulin production alone. Asclinical and research experience has become more extensive, it hasbecome apparent that some factor in addition to impairment of insulinsecretion is operative in diabetes. It is known that, while insulin isnormally deficient in diabetes, glucagon is normally present in excess.It is now believed that the presence of glucagon is at least asimportant a factor in diabetes as the absence of insulin.

The fact that a deficiency in insulin is normally accompanied by anexcess of glucagon has made it difficult to study the role of glucagonin diabetes. While it is easy to add extra quantities of a hormone suchas insulin, it has proved very difficult to lower the concentration ofglucagon. The discovery of somatostatin has facilitated research inrespect to the role of glucagon in diabetes. Somatostatin inhibits therelease of both insulin and glucagon. The role of somatostatin indiabetes research is detailed in an article appearing in Science, Vol.188, pp 920-923, May 30, 1975. However, there are several problems inrespect to the use of somatostatin as a treatment in diabetes.Somatostatin inhibits the release of insulin in addition to glucagon.Thus, the need for a peptide having a dissociated effect on theinhibition of release of insulin and glucagon has been recognized inconnection with diabetes treatment. The novel peptides of the presentinvention provide such dissociative effect. More particularly, certainof the peptides of the present invention are effective to inhibitsecretion of glucagon while having less effect on the inhibition ofsecretion of insulin.

The peptides of the invention provide the benefits of somatostatin andknown somatostatin analogs, but have fewer amino acid components, e.g. 8to 10 amino acid moieties as compared to 12 to 14 amino acid moietiesfor most known somatostatin analogs. Accordingly, the peptides of thepresent invention have significant economic advantage due to therelative ease of manufacture of these peptides.

The following examples illustrate various features of the presentinvention, but are intended to in no way limit the scope of theinvention which is defined in the appended claims.

EXAMPLE I

The peptides of the present invention were synthesized by solid phasetechniques, generally in accordance with the procedure described in U.S.Pat. No. 3,904,595. The synthesis was conducted in a stepwise manner onchloromethylated resin. The resin was composed of fine beads (20-70microns in diameter) of a synthetic resin prepared by copolymerizationof styrene with one to two percent divinylbenzene. The benzene rings inthe resin were chloromethylated in a Friedel-Crafts reaction withchloromethyl methyl ether and stannic chloride. The chlorine thusintroduced is a reactive benzyl chloride type of linkage. TheFriedel-Crafts reaction is continued until the resin contains 0.5 to 2millimoles of chlorine per gram of resin. In the further description ofthe synthesis of the peptides, the reagents used will be first describedby their chemical name with their common abbreviation in parenthesis.Thereafter, the reagent will be referred to by the common abbreviation.

A peptide having the structure:

    H-Cys-Phe-Phe-D-Trp-Lys-Phe-Phe-Cys-OH

was synthesized by the following solid phase methodology. Otherpeptides, described hereinafter were synthesized by a similar technique.

The tertiobutyloxycarbonyl-S-paramethoxybenzyl (Boc-SpOMe-Bzl)derivative of Cys was linked to the resin by any of three known methods;(1) reflux in ethanol in presence of triethyl amine, (2) Cesium salt ofthe Boc protected amino acid is kept at 50° C in dimethylformamide (DMF)overnight, (3) the potassium salt of the Boc-protected amino acid iskept at 80° C in dimethyl sulfoxide (DMSO) for 2 hours. Only onemilliequivalent of the protected Cys per milliequivalent of Cl on theresin is used.

Method (3) is described hereinbelow in more detail. To a slurry of theresin and the dissolved protected Cys in DMSO is added 0.9 mEq ofpotassium tertiobutoxide (KOtBut) per mEq of amino acid. The reactionmixture is exposed to air as little as possible so that no ambercoloration is observed. Reaction at 80° C for 2 hours yields a suitablesubstituted resin for synthesis of the peptides (approx. 0.2 mEq ofamino acid derivative per g of resin). After deprotection andneutralization, the peptide chain is built on resin. Deprotection,neutralization and addition of each amino acid is performed inaccordance with schedule I. N.sup.α -t-butyloxycarbonyl (Boc) derivativeof each amino acid is used. After deprotection of the first residue(i.e., SpOMe.Bzl.Cys) according to schedule I (steps 3 to 8 included),the N Boc derivative of Thr is next added along with a coupling agentwhich is dicyclohexylcarbodiimide (DCC) (step 9 of schedule I). The sidechain of Thr is protected with O-benzyl ether (OBzl). Benzyloxycarbonyl(Z) or benzyloxycarbonyl-2Cl [Z (2-Cl)] was used as the protecting groupfor the Lys side chain.

    ______________________________________                                        I. Schedule for coupling of amino acids in solid phase                        synthesis (5 - 10 g resin)                                                                                   Mix times                                      Step Reagents and operations   Min.                                           ______________________________________                                        1    CH.sub.2 Cl.sub.2 wash 80 ml (2 times)                                                                  3                                              2    Methanol (MeOH) wash 30 Ml (2 times)                                                                    3                                              3    CH.sub.2 Cl.sub.2 wash 80 ml (3 times)                                                                  3                                              4    50 percent trifluoroacetic acid (TFA)                                                                   10                                                  containing 5 percent 1,2-ethanedithiol                                        in CH.sub.2 Cl.sub.2 70 ml (2 times)                                     5    CH.sub.2 Cl.sub.2 wash 80 ml (2 times)                                                                  3                                              6    Triethylamine (Et.sub.3 N) 12.5 percent in                                                              5                                                   CH.sub.2 Cl.sub.2 70 ml (2 times)                                        7    MeOH wash 40 ml (2 times) 2                                              8    CH.sub.2 Cl.sub.2 wash 80 ml (3 times)                                                                  3                                              9    Boc-amino acid (10 mmoles) in 10 ml                                           DMF (1 times) and 30 ml CH.sub.2 Cl.sub.2 plus                                DCC (10 mmoles) in                                                            CH.sub.2 Cl.sub.2 (2 M)   30 to 120                                      10   MeOH wash 40 ml (2 times) 3                                              11   Et.sub.3 N 12.5 percent in CH.sub.2 Cl.sub.2 70 ml (2                                                   3imes)                                         12   MeOH wash 30 ml (2 times) 3                                              13   CH.sub.2 Cl.sub.2 wash 80 ml (2 times)                                                                  3                                              ______________________________________                                    

After step 13, an aliquot is taken for a ninhydrin test:

if the test is negative, go back to step 1 for coupling of the nextamino acid; if the test is positive or slightly positive, go back tosteps 9 through 13. Schedule I was used for coupling of each of theamino acids of the peptide to Cys.

Cleavage of the peptides from the resin (5 grams) and deprotection ofthe side chain protecting groups of the peptide was performed inhydrofluoric acid (75 ml) in the presence of anisole (8 ml). Afterelimination of hydrofluoric acid under high vacuum, the resin-peptidewas washed with ether.

The dried resin was immediately extracted with 25% acetic acid (150 ml)and diluted to 3000 ml with degassed H₂ O (N₂). The pH of the solutionwas adjusted to 6.6-7.0 with NH₄ 0H. The solution was titrated dropwiseunder stirring with potassium ferricyanide solution (1.g/500 ml H₂ O)until a permanent yellow color was observed. The solution sat for 10minutes and pH was adjusted to 5.0 with glacial acetic acid; Bio Rad AG3-X4A resin (100-200 mesh, chloride form, 10-15 g) was added to theturbid solution and stirred for 15 minutes. The solution was filteredover celite and applied successively onto two columns; (a) Bio Rad AG3-X4A resin chloride form (10 ml); (b) Bio Rex-70 resin (100 ml) cationform. The celite + resin cake was thoroughly washed with water (500 ml)which was applied onto columns (a) and (b) as a wash. The peptidematerial was then eluted from the Bio Rex-70 resin column with pyridine;acetic acid:water (30:4:66) or 50% acetic acid. Fractions werecollected; only the ones containing peptide (ninhydrin positive) werediluted with water and immediately lyophilized. 950 mg of crude creamcolored material was obtained. It was applied onto a Sephadex G-25 F gelcolumn (3 × 200 cm) equilibrated and eluted with 2 N acetic acid.

The elution pattern as observed at 280 nm showed one major symmetricalpeak. After lyophylization the center cut yielded 550 mg which weresubmitted to counter current distribution (solvent systemn-butanol:acetic acid:water, 4:1:5) 10 ml lower phase per tube. 100transfers were performed and the major peak was found in tubes 57-68.The compound (250 mg) appeared homogeneous on tlc.

The specific optical rotation was [α]²³ = -67.8 ± 2: (c=1 in 1% aceticacid). Amino acid analysis of this material showed the expected ratiofor the different amino acids.

Active esters can be used in solid phase synthesis and the classicalmethod of synthesis can also be used to prepare the peptides of theinvention.

In vitro Bioassay: The effects of the various peptides of the inventionwere tested in vitro on the secretion of growth hormone by primarycultures of enzymatically dissociated rat anterior pituitary cells bythe method of Vale et al., Endocrinology 91: p 562-571 (1972). The assayis made by treating pituitary glands removed from rats to separate cellstherefrom. The cells are placed in culture dishes in Dulbecco's ModifiedEagle Medium (Dulbecco et al., Virology, Vol. 8, p. 396, 1949). Carbondioxide gas and oxygen are supplied to the cell cultures which aremaintained at 37° C for 4-5 days prior to use in the assay. Followingmedia changes, cell cultures are incubated for a period of 4 hours andparticular somatostatin peptides are added thereto. Radioimmunoassayanalysis is used to determine the rate of growth hormone secretion whichis expressed in nanograms per hour.

An investigation of the effect of somatostatin, dihydrosomatostatin, (ascontrols) and the peptides of the invention to inhibit the release ofglucagon and insulin was made as follows:

In vivo Bioassay: Male Sprague-Dawley-CD rats weighing 180-200 g housedin temperature and humidity controlled quarters with 14h light and 10hdark (light 0700-21100) were used in all experiments. Animals were fed astandard ration and tap water ad libitum. Experiments were carried outat least 5 days after arrival of rats from the supplier between thehours 1400 to 1600. After ether anesthesia, peptides or saline wereadministered in a volume of 0.2 ml. via the external jugular vein.Animals remained anesthetized until the time of blood collection fromthe portal vein. The blood samples were placed into chilled tubescontaining 10 mg EDTA and 50 μl of M Benzamidine per ml of blood.

Plasma was stored at -20° C for insulin and glucagon determinations.Insulin levels were determined by the method of Herbert et al, J. Chem.Endocr. Metab. 25:1375, 1956, utilizing porcine insulin antisera and(125I) iodinated insulin tracer. Human insulin standard was obtainedfrom Schwarz-Mann, Orangeburg, New York. Glucagon was determined by themethod of Faloona and Unger, in Jaffe et al ed., Methods of HormoneRadioimmunoassay, Academic Press, New York, 1974, p. 317, utilizingglucagon antisera 30K. Cellulose was determined by the glucose oxidasemethod, utilizing a Beckman Glucose Analyzer.

GH determinations were performed on tissue culture media utilizing thefollowing reagents: NIAMDD rat GH standard (GH-RP-1), NIAMDD monkeyanti-rat GH (GH-Serum-3), and highly purified rat GH for iodination.

All experiments were carried on in a randomized block design. Followinganalysis of variance difference between treatments were determined bythe multiple range tests of Dunnett and Duncan. Potency values werecalculated from four or six point bioassays.

Various peptides in accordance with the invention were prepared inaccordance with the solid phase methodology described above. Thecomposition of the peptides is reported hereinbelow in Table I. Table Ialso sets forth the percent effectiveness of the peptide for inhibitingsecretion of growth hormone (GH), insulin and glucagon, withsomatostatin taken as the base.

                                      TABLE I                                     __________________________________________________________________________                           Growth                                                 Somatostatin (control) Hormone                                                                            Insulin                                                                           Glucagon                                      Peptides of Invention  100  100 100                                           __________________________________________________________________________    R.sub.1                                                                           R.sub.2                                                                           R.sub.3                                                                          R.sub.4                                                                           R.sub.5                                                        desR.sub.1                                                                        D-Trp                                                                             Phe                                                                              desR.sub.4                                                                        desR.sub.5                                                                            6    80  100                                           desR.sub.1                                                                        D-Trp                                                                             Phe                                                                              desR.sub.4                                                                        desR.sub.5 (D-Cys).sup.11                                                             14   100 100                                           desR.sub.1                                                                        D-Trp                                                                             Phe                                                                              Thr desR.sub.5                                                                            35   10-100                                                                            100                                           Asn D-Trp                                                                             Phe                                                                              desR.sub.4                                                                        desR.sub.5                                                                            <1   100 <1                                            Asn Trp Phe                                                                              Thr desR.sub.5                                                                            3    100 <1                                            Asn D-Trp                                                                             Phe                                                                              Thr desR.sub.5                                                                            10   200 10-100                                        __________________________________________________________________________

What is claimed is:
 1. A peptide selected from those of the formulae:##STR3##

    Cys-R.sub.1 -Phe-Phe-R.sub.2 -Lys-R.sub.3 -Phe-R.sub.4 -R.sub.5 -Cys II

    x-cys-(X.sup.1)-R.sub.1 -Phe-Phe-R.sub.2 -Lys(X.sup.2)-R.sub.3 (X.sup.3)-Phe-R.sub.4 (X.sup.4)-R.sub.5 (X.sup.5)-Cys (X.sup.6)-R.sub.6 III

wherein R₁ is selected from the group consisting of Asn and desR₁ ; R₂is selected from the group consisting of Trp and D-Trp; R₃ is selectedfrom the group consisting of Phe and Thr; R₄ is selected from groupconsisting of Thr and desR₄ ; R₅ is selected from the group consistingof Ser, Phe and desR₅, provided that at least one of R₁, R₄ and R₅ isdeleted; X is selected from the group consisting of H, and analpha-amino protecting group; X¹ and X⁶ are selected from the groupconsisting of H and a protecting group for Cys selected fromS-p-methoxybenzyl, S-acetamidomethyl, S-trityl and S-benzyl; X² isselected from the group consisting of H and a side chain aminoprotecting group; X³, X⁴ and X⁵ are selected from the group consistingof H and a hydroxyl protecting group selected from the group consistingof acetyl, benzoyl, tert-butyl, trityl, benzyl and benzyloxycarbonyl;with the proviso that at least one of X, X¹, X², X³, X⁴, X⁵, and X⁶ isother than hydrogen; and R₆ is selected from the group consisting ofhydroxy, methoxy, and an anchoring bond used in solid phase synthesislinked to a solid resin support selected from the group consisting of--O---CH₂ -polystyrene resin support and O--CH₂ -benzylpolystyrene resinsupport.
 2. A peptide in accordance with claim 1 wherein R₁ is des R₁,R₂ is D-Trp, R₃ is Phe, R₄ is desR₄ and R₅ is desR₅.
 3. A peptide inaccordance with claim 1 wherein R₁ is desR₁, R₂ is D-Trp, R₃ is Phe, R₄is DesR₄, R₅ is desR₅ and D-Cys is substituted for Cys¹¹.
 4. A peptidein accordance with claim 1 wherein R₁ is desR₁, R₂ is D-Trp, R₃ is Phe,R₄ is desR₄ and R₅ is desR₅.
 5. A peptide in accordance with claim 1wherein R₁ is Asn, R₂ is D-Trp, R₃ is Phe, R₄ is desR₄ and R₅ is desR₅.6. A peptide in accordance with claim 1 wherein R₁ is Asn, R₂ is Trp, R₃is Phe, R₄ is Thr and R₅ is desR₅.
 7. A peptide in accordance with claim1 wherein R₁ is Asn, R₂ is D-Trp, R₃ is Phe, R₄ is Thr and R₅ is des R₅.